Interfacing capillary electrophoresis to a mass spectrometer via an impactor spray ionization source

ABSTRACT

A mass spectrometer is disclosed comprising a separation device arranged and adapted to emit an eluent over a period of time. The separation device preferably comprises a Capillary Electrophoresis (“CE”) separation device. The mass spectrometer further comprises a nebuliser and a target. Eluent emitted by the separation device is nebulised, in use, by the nebuliser wherein a stream of analyte droplets are directed to impact upon the target so as to ionise the analyte to form a plurality of analyte ions.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.14/367,647 filed 20 Jun. 2014, which is the National Stage ofInternational Application No. PCT/GB2012/053258, filed 21 Dec. 2012,which claims priority from and the benefit of U.S. Provisional PatentApplication Ser. No. 61/580,558 filed on 27 Dec. 2011, U.S. ProvisionalPatent Application Ser. No. 61/601,827 filed on 22 Feb. 2012, U.S.Provisional Patent Application Ser. No. 61/718,836 filed on 26 Oct.2012, United Kingdom Patent Application No. 1122218.9 filed on 23 Dec.2011, United Kingdom Patent Application No. 1202892.4 filed on 21 Feb.2012 and United Kingdom Patent Application No. 1219217.5 filed on 25Oct. 2012. The entire contents of these applications are incorporatedherein by reference.

BACKGROUND TO THE PRESENT INVENTION

Capillary Electrophoresis (CE) is a separation technique where a highvoltage is applied to the sample inlet end of a glass capillary columnand a lower voltage, or voltage of opposite polarity, is applied to theoutlet end of the capillary. Analytes elute from the column at a ratethat is determined by a combination of electroosmotic flow and theelectrophoretic mobility of the analytes. Since the electroosmotic flowvelocity can exceed the electrophoretic drift velocity of ions, it ispossible to analyse both positive and negative ions in the samechromatographic separation. In such circumstances, the elution order canbe generalized as multiply charged positive ions emerging first,followed by singly charged positive ions, followed by neutral analytes,followed by singly charged negative ions and finally followed bymultiply charged negative ions. Commonly used CE detectors such as UVand fluorescence devices can analyse both ion polarities in a singlechromatographic run.

However, when interfacing CE to mass spectrometry via an Electrosprayionization source, the column outlet is located at the Electrosprayprobe tip which in turn, is biased to typically 3 kV via a separate highvoltage supply and a potential divider circuit. The analysis of positiveand negative ions requires ESI tip voltages of +3 kV and −3 kVrespectively. This precludes the use of fast positive/negative switchingin a single chromatographic run since this would affect the total CEvoltage and hence electroosmotic and electrophoretic flows.

Another disadvantage of the conventional arrangement is that biasing theESI tip requires the additional cost of an ESI power supply circuit.Furthermore, such an arrangement imposes limitations on bufferconcentration and ESI voltage stability.

It is therefore desired to provide an improved mass spectrometer.

SUMMARY OF THE PRESENT INVENTION

According to an aspect of the present invention there is provided a massspectrometer comprising:

a separation device arranged and adapted to emit an eluent over a periodof time, wherein the separation device comprises either: (i) a CapillaryElectrophoresis (“CE”) separation device; (ii) a CapillaryElectrochromatography (“CEC”) separation device; (iii) a substantiallyrigid ceramic-based multilayer microfluidic substrate (“ceramic tile”)separation device; or (iv) a supercritical fluid chromatographyseparation device;

a nebuliser and;

a target;

wherein the eluent emitted by the separation device is nebulised, inuse, by the nebuliser wherein a stream of analyte droplets are directedto impact upon the target so as to ionise the analyte to form aplurality of analyte ions.

The present invention is particularly advantageous in that a CapillaryElectrophoresis (“CE”) separation device and other types of separationdevice may be arranged to emit an eluent which is then nebulised so thata resulting stream of droplets is then ionised upon impacting a target.Interfacing a CE separation device to an impact ionisation sourceaccording to the present invention is particularly advantageous sincethe conventional teaching is to interface a CE separation device to anElectrospray ionisation source. However, this involves maintaining theElectrospray probe tip at +3 kV and being able rapidly to switch theprobe tip to −3 kV in order to produce negative ions. It is problematicto maintain the Electrospray probe tip at 3 kV when coupled to a CEseparation device and it is not possible to rapidly switch the voltageof the probe tip as this would affect the total CE voltage and hence theelectroosmotic and electrophoretic flows.

The present invention, therefore, enables the probe tip to be maintainede.g. at ground potential and avoids the expense and added complicationof requiring a fast switching high voltage power supply for the probe.

The present invention wherein a CE separation device is coupled with animpact ionisation ion source is, therefore, particularly advantageouscompared to conventional arrangement wherein a CE separation device isinterfaced with a high voltage Electrospray ionisation ion source.

According to a preferred embodiment of the present invention the liquidflow from the outlet of a capillary electrophoresis column is connectedto the inner capillary of a grounded, tri-axial, pneumatic nebuliserprobe. A flow of make-up solution is added to the second concentriccapillary which mixes with the flow from the inner capillary at theprobe tip. The resulting liquid stream is converted into a nebulisedspray via a concentric flow of high velocity gas from a third concentriccapillary. A small impactor target is preferably positioned inrelatively close proximity to the nebuliser tip to define an impact zoneand to ionize the incoming high velocity droplet stream. The resultingions and charged droplets are sampled by a first vacuum stage of a massspectrometer.

The ionizing high voltage is decoupled from the probe tip and a groundedprobe assembly can advantageously be utilised which acts as a stablereference for the applied CE voltage. This is particularly advantageouscompared to conventional arrangements.

The nebuliser probe tip is preferably held at (or relatively close to)ground potential whilst any high voltage for ionization is preferablyheld on an impactor target that is positioned a short distance from thetip. This arrangement eliminates the problems described above andenables the use of fast polarity switching of the impactor target toanalyse both positive and negative ions in a single CE/MS run.

The impactor spray source may generally operate at liquid flow rates ≦1μL/min. However, since the electroosmotic flow associated with a CEcolumn is extremely low (<<1 μL/min), the impactor spray nebuliser probeis preferably constructed with a triaxial probe arrangement thatincreases the total liquid flow rate. An inner capillary is preferablyconnected to the outlet of a CE capillary column. The inner capillary ispreferably surrounded by a second concentric capillary that preferablydelivers a make-up flow of liquid that mixes with the liquid flow fromthe CE column. The second capillary is preferably surrounded by a thirdconcentric capillary which preferably delivers high velocity nitrogengas to nebulise the resulting liquid flows from the other twocapillaries. All three capillaries in the tri-axial arrangement arepreferably maintained at ground potential. An impactor target ispreferably held at a relatively high potential and is preferablypositioned in relatively close proximity to the probe tip.

According to a preferred embodiment the liquid flow from the outlet of acapillary electrophoresis column is connected to the inner capillary ofgrounded, preferably tri-axial, pneumatic nebuliser probe. A flow ofmake-up solution is added to the second concentric capillary which mixeswith the flow from the inner capillary at the probe tip. The resultingliquid stream is converted into a nebulised spray via a concentric flowof high velocity gas from a third concentric capillary. A small impactortarget is positioned in close proximity to the nebuliser tip to definean impact zone and to ionize the incoming high velocity droplet stream.The resulting ions and charged droplets are sampled by the first vacuumstage of a mass spectrometer.

The preferred embodiment alleviates interfacing problems by decouplingthe ionizing high voltage from the probe tip and instead preferablyutilizing a grounded probe assembly which acts as a stable reference forthe applied CE voltage. In Impactor Spray ionization, the nebuliserprobe tip is preferably held at ground potential whilst any high voltagefor ionization is preferably held on an impactor target that ispreferably positioned a short distance from the tip. This arrangementeliminates the problems described above and enables the use of fastpolarity switching of the impactor target to analyse both positive andnegative ions in a single CE/MS run.

High voltage ESI nebuliser probes can complicate interfacing MS sourceswith high voltage chromatography techniques such as CE and CEC. Thepreferred embodiment solves the problem of creating ionization withoutapplying a high voltage directly to the nebuliser probe.

The separation device preferably comprises a Capillary Electrophoresis(“CE”) separation device wherein an inlet end of the CapillaryElectrophoresis separation device is maintained at a first potential andan outlet end of the Capillary Electrophoresis separation device ismaintained at a second potential.

The separation device preferably comprises or is coupled to a firsttube.

The first tube preferably comprises a capillary tube.

The exit of the first tube is preferably maintained, in use, at apotential (i) −5 to −4 kV; (ii) −4 to −3 kV; (iii) −3 to −2 kV; (iv) −2to −1 kV; (v) −1000 to −900 V; (vi) −900 to −800 V; (vii) −800 to −700V; (viii) −700 to −600 V; (ix) −600 to −500 V; (x) −500 to −400 V; (xi)−400 to −300 V; (xii) −300 to −200 V; (xiii) −200 to −100 V; (xiv) −100to −90 V; (xv) −90 to −80 V; (xvi) −80 to −70 V; (xvii) −70 to −60 V;(xviii) −60 to −50 V; (xix) −50 to −40 V; (xx) −40 to −30 V; (xxi) −30to −20 V; (xxii) −20 to −10 V; (xxiii) −10 to 0V; (xxiv) 0-10 V; (xxv)10-20 V; (xxvi) 20-30 V; (xxvii) 30-40V; (xxviii) 40-50 V; (xxix) 50-60V; (xxx) 60-70 V; (xxxi) 70-80 V; (xxxii) 80-90 V; (xxxiii) 90-100 V;(xxxiv) 100-200 V; (xxxv) 200-300 V; (xxxvi) 300-400 V; (xxxvii) 400-500V; (xxxviii) 500-600 V; (xxxix) 600-700 V; (xl) 700-800 V; (xli) 800-900V; (xlii) 900-1000 V; (xliii) 1-2 kV; (xliv) 2-3 kV; (xlv) 3-4 kV; and(xlvi) 4-5 kV.

According to a less preferred embodiment the exit of the first tube maybe maintained at a potential <−5 kV or >5 kV.

The first tube is preferably surrounded by a second tube which isarranged and adapted to provide a flow of liquid which mixes with theeluent emerging from the exit of the first tube.

The second tube preferably comprises a capillary tube.

The ends of the first and second tubes may be either: (i) flush orparallel with each other; or (ii) protruded, recessed or non-parallelrelative to each other.

The mass spectrometer preferably comprises a third tube which maysurround the second tube and which is arranged and adapted to provide astream of gas to the exit of the first tube and/or the second tube.

The third tube preferably comprises a capillary tube.

The third tube preferably surrounds the second tube and/or is concentricwith the first and second tubes.

According to an embodiment the ends of the first, second and third tubesmay be either: (i) flush or parallel with each other; or (ii) protruded,recessed or non-parallel relative to each other.

According to an alternative embodiment the third tube may benon-concentric with the first and the second tubes.

The mass spectrometer preferably further comprises a heater which isarranged and adapted to supply a heated stream of gas to heat dropletsemerging from the first tube and/or the second tube.

The target is preferably arranged <10 mm, <9 mm, <8 mm, <7 mm, <6 mm, <5mm, <4 mm, <3 mm or <2 mm from the exit of the nebuliser.

The target is preferably maintained, in use, at a potential (i) −5 to −4kV; (ii) −4 to −3 kV; (iii) −3 to −2 kV; (iv) −2 to −1 kV; (v) −1000 to−900 V; (vi) −900 to −800 V; (vii) −800 to −700 V; (viii) −700 to −600V; (ix) −600 to −500 V; (x) −500 to −400 V; (xi) −400 to −300 V; (xii)−300 to −200 V; (xiii) −200 to −100 V; (xiv) −100 to −90 V; (xv) −90 to−80 V; (xvi) −80 to −70 V; (xvii) −70 to −60 V; (xviii) −60 to −50 V;(xix) −50 to −40 V; (xx) −40 to −30 V; (xxi) −30 to −20 V; (xxii) −20 to−10 V; (xxiii) −10 to 0V; (xxiv) 0-10 V; (xxv) 10-20 V; (xxvi) 20-30 V;(xxvii) 30-40V; (xxviii) 40-50 V; (xxix) 50-60 V; (xxx) 60-70 V; (xxxi)70-80 V; (xxxii) 80-90 V; (xxxiii) 90-100 V; (xxxiv) 100-200 V; (xxxv)200-300 V; (xxxvi) 300-400 V; (xxxvii) 400-500 V; (xxxviii) 500-600 V;(xxxix) 600-700 V; (xl) 700-800 V; (xli) 800-900 V; (xlii) 900-1000 V;(xliii) 1-2 kV; (xliv) 2-3 kV; (xlv) 3-4 kV; and (xlvi) 4-5 kV.

According to a less preferred embodiment the target may be maintained ata potential <−5 kV or >5 kV.

The mass spectrometer further comprises a control system, wherein thecontrol system is arranged and adapted either: (i) to switch thepolarity of said target during a single experimental run; or (ii) torepeatedly switch the polarity of the target during a singleexperimental run.

According to an embodiment the control system may be arranged andadapted to repeatedly switch the polarity of said target every 0-10 ms,10-20 ms, 20-30 ms, 30-40 ms, 40-50 ms, 50-60 ms, 60-70 ms, 70-80 ms,80-90 ms, 90-100 ms, 100-200 ms, 200-300 ms, 300-400 ms, 400-500 ms,500-600 ms, 600-700 ms, 700-800 ms, 800-900 ms, 900-1000 ms, 1-2 s, 2-3s, 3-4 s or 4-5 s.

According to another embodiment the control system may utilise retentiontime switching. According to an embodiment the polarity of the targetmay be repeatedly switched once every 0-1 mins, 1-2 mins, 2-3 mins, 3-4mins, 4-5 mins, 5-6 mins, 6-7 mins, 7-8 mins, 8-9 mins, 9-10 mins or >10mins.

The mass spectrometer preferably further comprises an enclosureenclosing the nebuliser, the target and an ion inlet device which leadsto a first vacuum stage of the mass spectrometer.

According to an embodiment the ion inlet device may comprise an ionorifice, an ion inlet cone, an ion inlet capillary, an ion inlet heatedcapillary, an ion tunnel, an ion mobility spectrometer or separator, adifferential ion mobility spectrometer, a Field Asymmetric Ion MobilitySpectrometer (“FAIMS”) device or other ion inlet.

The exit of the first tube preferably has a diameter D and the spray ofanalyte droplets is preferably arranged to impact on an impact zone ofthe target.

The impact zone preferably has a maximum dimension of x and wherein theratio x/D is in the range <2, 2-5, 5-10, 10-15, 15-20, 20-25, 25-30,30-35, 35-40 or >40.

The impact zone preferably has an area selected from the groupconsisting of: (i)<0.01 mm²; (ii) 0.01-0.10 mm²; (iii) 0.10-0.20 mm²;(iv) 0.20-0.30 mm²; (v) 0.30-0.40 mm²; (vi) 0.40-0.50 mm²; (vii)0.50-0.60 mm²; (viii) 0.60-0.70 mm²; (ix) 0.70-0.80 mm²; (x) 0.80-0.90mm²; (xi) 0.90-1.00 mm²; (xii) 1.00-1.10 mm²; (xiii) 1.10-1.20 mm²;(xiv) 1.20-1.30 mm²; (xv) 1.30-1.40 mm²; (xvi) 1.40-1.50 mm²; (xvii)1.50-1.60 mm²; (xviii) 1.60-1.70 mm²; (xix) 1.70-1.80 mm²; (xx)1.80-1.90 mm²; (xxi) 1.90-2.00 mm²; (xxii) 2.00-2.10 mm²; (xxiii)2.10-2.20 mm²; (xxiv) 2.20-2.30 mm²; (xxv) 2.30-2.40 mm²; (xxvi)2.40-2.50 mm²; (xxvii) 2.50-2.60 mm²; (xxviii) 2.60-2.70 mm²; (xxix)2.70-2.80 mm²; (xxx) 2.80-2.90 mm²; (xxxi) 2.90-3.00 mm²; (xxxii)3.00-3.10 mm²; (xxxiii) 3.10-3.20 mm²; (xxxiv) 3.20-3.30 mm²; (xxxv)3.30-3.40 mm²; (xxxvi) 3.40-3.50 mm²; (xxxvii) 3.50-3.60 mm²; (xxxviii)3.60-3.70 mm²; (xxxix) 3.70-3.80 mm²; (xl) 3.80-3.90 mm²; and (xli)3.90-4.00 mm².

The target is preferably located at a first distance X₁ in a firstdirection from an ion inlet device which leads to a first vacuum stageof the mass spectrometer and at a second distance Z₁ in a seconddirection from the ion inlet device, wherein the second direction isorthogonal to the first direction and wherein:

(i) X₁ is selected from the group consisting of: (i) 0-1 mm; (ii) 1-2mm; (iii) 2-3 mm; (iv) 3-4 mm; (v) 4-5 mm; (vi) 5-6 mm; (vii) 6-7 mm;(viii) 7-8 mm; (ix) 8-9 mm; (x) 9-10 mm; and (xi) >10 mm; and/or

(ii) Z₁ is selected from the group consisting of: (i) 0-1 mm; (ii) 1-2mm; (iii) 2-3 mm; (iv) 3-4 mm; (v) 4-5 mm; (vi) 5-6 mm; (vii) 6-7 mm;(viii) 7-8 mm; (ix) 8-9 mm; (x) 9-10 mm; and (xi) >10 mm.

The target is preferably positioned so as to deflect the stream ofanalyte droplets and/or the plurality of analyte ions towards an ioninlet device of the mass spectrometer.

According to an embodiment the ion inlet device may comprise an ionorifice, an ion inlet cone, an ion inlet capillary, an ion inlet heatedcapillary, an ion tunnel, an ion mobility spectrometer or separator, adifferential ion mobility spectrometer, a Field Asymmetric Ion MobilitySpectrometer (“FAIMS”) device or other ion inlet.

The target is preferably positioned upstream of an ion inlet device ofthe mass spectrometer so that ions are deflected towards the directionof the ion inlet device.

The target preferably comprises either: (i) a rod; or (ii) a pin havinga taper cone;

wherein the stream of analyte droplets is arranged to impact the rod orthe taper cone of the pin either: (i) directly on the centerline of therod or pin; or (ii) on the side of the rod or the taper cone which facestowards or away from an ion inlet orifice of the mass spectrometer.

The ion source preferably comprises an Atmospheric Pressure Ionisation(“API”) ion source.

The target preferably comprises a stainless steel target, a metal, gold,a non-metallic substance, a semiconductor, a metal or other substancewith a carbide coating, an insulator or a ceramic.

According to an embodiment the target may comprise a plurality of platesso that droplets from the nebuliser cascade upon a plurality of targetplates and/or wherein the target is arranged to have multiple impactpoints so that droplets are ionised by multiple glancing deflections.

According to an embodiment the target comprises one or more mesh or gridtargets.

A grid or mesh target having a grid or mesh impaction surface has beenfound to be particularly advantageous compared with using a pin targetsince utilising a grid or mesh target solves the problem of positionaldependence which may otherwise be experienced when using a solid pin asthe target.

The one or more mesh or grid targets preferably comprise one or morewire mesh or grid targets.

The wire mesh or grid target preferably comprise wire having a diameterselected from the group consisting of: (i)<50 μm; (ii) 50-100 μm; (iii)100-150 μm; (iv) 150-200 μm; (v) 200-250 μm; (vi) 250-300 μm; (vii)300-350 μm; (viii) 350-400 μm; (ix) 400-450 μm; (x) 450-500 μm; (xi)500-550 μm; (xii) 550-600 μm; (xiii) 600-650 μm; (xiv) 650-700 μm; (xv)700-750 μm; (xvi) 750-800 μm; (xvii) 800-850 μm; (xviii) 850-900 μm;(xix) 900-950 μm; (xx) 950-1000 μm; and (xxi) >1 mm.

The mesh or grid preferably has a spacing selected from the groupconsisting of: (i)<50 μm; (ii) 50-100 μm; (iii) 100-150 μm; (iv) 150-200μm; (v) 200-250 μm; (vi) 250-300 μm; (vii) 300-350 μm; (viii) 350-400μm; (ix) 400-450 μm; (x) 450-500 μm; (xi) 500-550 μm; (xii) 550-600 μm;(xiii) 600-650 μm; (xiv) 650-700 μm; (xv) 700-750 μm; (xvi) 750-800 μm;(xvii) 800-850 μm; (xviii) 850-900 μm; (xix) 900-950 μm; (xx) 950-1000μm; and (xxi) >1 mm.

The one or more mesh or grid targets are preferably arranged in a planewhich is either: (i) substantially perpendicular to a spray axis of theone or more nebulisers; or (ii) inclined at an angle <90° to a sprayaxis of the one or more nebulisers.

The one or more mesh or grid targets preferably provide multiple impactzones.

The one or more mesh or grid targets preferably comprise a 1-dimensionalor a 2-dimensional array of interstices or openings.

The one or more mesh or grid targets preferably comprise a plurality oflayers.

One or more of the layers preferably comprises a mesh or grid.

The plurality of layers preferably comprise layers having substantiallythe same or substantially different mesh sizes.

According to an embodiment the mass spectrometer further comprises avibration device arranged and adapted to cause the target to vibrate.

The use of piezoelectric vibration applied to the impactor bar or targetis particularly advantageous in that vibrating the target aids in thereduction of resultant secondary droplets through surface disruption.The use of piezoelectric vibration is also particularly advantageous inthat it also reduces liquid beading.

The vibration source is preferably arranged and adapted to cause thetarget to vibrate in order to reduce the size of resultant secondarydroplets through surface disruption.

The vibration source preferably comprises a piezo-electric vibrationsource.

The vibration source is preferably arranged and adapted to vibrate thetarget at a frequency f selected from the group consisting of: (i)<1kHz; (ii) 1-2 kHz; (iii) 2-3 kHz; (iv) 3-4 kHz; (v) 4-5 kHz; (vi) 5-6kHz; (vii) 6-7 kHz; (viii) 7-8 kHz; (ix) 8-9 kHz; (x) 9-10 kHz; (xi)10-11 kHz; (xii) 11-12 kHz; (xiii) 12-13 kHz; (xiv) 13-14 kHz; (xv)14-15 kHz; (xvi) 15-16 kHz; (xvii) 16-17 kHz; (xviii) 17-18 kHz; (xix)18-19 kHz; (xx) 19-20 kHz; and (xxi) >20 kHz.

According to an embodiment the mass spectrometer preferably furthercomprises a first device arranged and adapted to rotate and/or translatethe target.

As will be understood by those skilled in the art the positioning of thetarget is important in order to obtain an acceptable level of signalintensity when generating ions by impacting high velocity droplets ontothe target. According to a particularly preferred embodiment causing thetarget to rotate (e.g. on an eccentric path) relative to the spray ofhigh velocity droplets enables an average more stable signal intensityto be realised. As a result, the overall or average ion signal can bestabilised and is less susceptible to wide variations in the intensityof analyte ions generated which depends upon the precise position of thetarget relative to the high velocity spray of droplets.

The target preferably comprises a pin or rod.

The target preferably has a first central longitudinal axis and thefirst device is arranged and adapted to rotate the target about a secondaxis which is displaced or offset from the first axis.

The first device is preferably arranged and adapted to cause the targetto rotate, in use, about or on a substantially eccentric or non-circularpath.

The first device is preferably arranged and adapted to rotate the targetat a rate of: (i)<1 rev/s; (ii) 1-2 rev/s; (iii) 2-3 rev/s; (iv) 3-4rev/s; (v) 4-5 rev/s; (vi) 5-6 rev/s; (vii) 6-7 rev/s; (viii) 7-8 rev/s;(ix) 8-9 rev/s; (x) 9-10 rev/s; (xi) >10 rev/s; (xii)<1 rpm; (xiii) 1-5rpm; (xiv) 5-10 rpm; (xv) 10-15 rpm; (xvi) 15-20 rpm; (xvii) 20-25 rpm;(xviii) 25-30 rpm; (xix) 30-35 rpm; (xx) 35-40 rpm; (xxi) 40-45 rpm;(xxii) 45-50 rpm; (xxiii) 50-60 rpm; (xxiv) 60-70 rpm: (xxv) 70-80 rpm;(xxvi) 80-90 rpm; (xxvii) 90-100 rpm; (xxviii) 100-150 rpm; (xxix)150-200 rpm; (xxx) 200-250 rpm; and (xxxi) >250 rpm.

The first device is preferably arranged and adapted to rotate the targetsubstantially continuously.

The first device is preferably arranged and adapted to rotate the targetsubstantially continuously for at least a period T, wherein T isselected from the group consisting of: (i)<1 s; (ii) 1-5 s; (iii) 5-10s; (iv) 10-15 s; (v) 15-20 s; (vi) 20-25 s; (vii) 25-30 s; (viii) 30-35s; (ix) 35-40 s; (x) 40-45 s; (xi) 45-50 s; (xii) 50-55 s; (xiii) 55-60s; and (xiv) >60 s.

The mass spectrometer preferably comprises a control system arranged andadapted to monitor an analyte signal as a function of or in respect ofthe position of the target.

The control system is preferably arranged and adapted to cause a deviceto rotate and/or translate the target to a desired position in order tooptimise an analyte ion signal or to otherwise control the intensity ofanalyte ions.

The control system is preferably arranged and adapted to cause a deviceto rotate and/or translate the target between a plurality of desiredpositions in order to vary or control the intensity of analyte ions.

According to an aspect of the present invention there is provided amethod of mass spectrometry comprising:

providing a separation device arranged and adapted to emit an eluentover a period of time, wherein the separation device comprises either:(i) a Capillary Electrophoresis (“CE”) separation device; (ii) aCapillary Electrochromatography (“CEC”) separation device; (iii) asubstantially rigid ceramic-based multilayer microfluidic substrate(“ceramic tile”) separation device; or (iv) a supercritical fluidchromatography separation device;

providing a target; and

nebulising the eluent emitted by the separation device wherein a streamof analyte droplets are directed to impact upon the target so as toionise the analyte to form a plurality of analyte ions.

According to an embodiment the mass spectrometer may further comprise:

(a) an additional ion source selected from the group consisting of: (i)an Electrospray ionisation (“ESI”) ion source; (ii) an AtmosphericPressure Photo Ionisation (“APPI”) ion source; (iii) an AtmosphericPressure Chemical Ionisation (“APCI”) ion source; (iv) a Matrix AssistedLaser Desorption Ionisation (“MALDI”) ion source; (v) a Laser DesorptionIonisation (“LDI”) ion source; (vi) an Atmospheric Pressure Ionisation(“API”) ion source; (vii) a Desorption Ionisation on Silicon (“DIOS”)ion source; (viii) an Electron Impact (“EI”) ion source; (ix) a ChemicalIonisation (“CI”) ion source; (x) a Field Ionisation (“FI”) ion source;(xi) a Field Desorption (“FD”) ion source; (xii) an Inductively CoupledPlasma (“ICP”) ion source; (xiii) a Fast Atom Bombardment (“FAB”) ionsource; (xiv) a Liquid Secondary Ion Mass Spectrometry (“LSIMS”) ionsource; (xv) a Desorption Electrospray Ionisation (“DESI”) ion source;(xvi) a Nickel-63 radioactive ion source; (xvii) an Atmospheric PressureMatrix Assisted Laser Desorption Ionisation ion source; (xviii) aThermospray ion source; (xix) an Atmospheric Sampling Glow DischargeIonisation (“ASGDI”) ion source; (xx) a Glow Discharge (“GD”) ionsource; and (xxi) an Impactor ion source; and/or

(b) one or more continuous or pulsed ion sources; and/or

(c) one or more ion guides; and/or

(d) one or more ion mobility separation devices and/or one or more FieldAsymmetric Ion Mobility Spectrometer devices; and/or

(e) one or more ion traps or one or more ion trapping regions; and/or

(f) one or more collision, fragmentation or reaction cells selected fromthe group consisting of: (i) a Collisional Induced Dissociation (“CID”)fragmentation device; (ii) a Surface Induced Dissociation (“SID”)fragmentation device; (iii) an Electron Transfer Dissociation (“ETD”)fragmentation device; (iv) an Electron Capture Dissociation (“ECD”)fragmentation device; (v) an Electron Collision or Impact Dissociationfragmentation device; (vi) a Photo Induced Dissociation (“PID”)fragmentation device; (vii) a Laser Induced Dissociation fragmentationdevice; (viii) an infrared radiation induced dissociation device; (ix)an ultraviolet radiation induced dissociation device; (x) anozzle-skimmer interface fragmentation device; (xi) an in-sourcefragmentation device; (xii) an in-source Collision Induced Dissociationfragmentation device; (xiii) a thermal or temperature sourcefragmentation device; (xiv) an electric field induced fragmentationdevice; (xv) a magnetic field induced fragmentation device; (xvi) anenzyme digestion or enzyme degradation fragmentation device; (xvii) anion-ion reaction fragmentation device; (xviii) an ion-molecule reactionfragmentation device; (xix) an ion-atom reaction fragmentation device;(xx) an ion-metastable ion reaction fragmentation device; (xxi) anion-metastable molecule reaction fragmentation device; (xxii) anion-metastable atom reaction fragmentation device; (xxiii) an ion-ionreaction device for reacting ions to form adduct or product ions; (xxiv)an ion-molecule reaction device for reacting ions to form adduct orproduct ions; (xxv) an ion-atom reaction device for reacting ions toform adduct or product ions; (xxvi) an ion-metastable ion reactiondevice for reacting ions to form adduct or product ions; (xxvii) anion-metastable molecule reaction device for reacting ions to form adductor product ions; (xxviii) an ion-metastable atom reaction device forreacting ions to form adduct or product ions; and (xxix) an ElectronIonisation Dissociation (“EID”) fragmentation device; and/or

(g) a mass analyser selected from the group consisting of: (i) aquadrupole mass analyser; (ii) a 2D or linear quadrupole mass analyser;(iii) a Paul or 3D quadrupole mass analyser; (iv) a Penning trap massanalyser; (v) an ion trap mass analyser; (vi) a magnetic sector massanalyser; (vii) Ion Cyclotron Resonance (“ICR”) mass analyser; (viii) aFourier Transform Ion Cyclotron Resonance (“FTICR”) mass analyser; (ix)an electrostatic or orbitrap mass analyser; (x) a Fourier Transformelectrostatic or orbitrap mass analyser; (xi) a Fourier Transform massanalyser; (xii) a Time of Flight mass analyser; (xiii) an orthogonalacceleration Time of Flight mass analyser; and (xiv) a linearacceleration Time of Flight mass analyser; and/or

(h) one or more energy analysers or electrostatic energy analysers;and/or

(i) one or more ion detectors; and/or

(j) one or more mass filters selected from the group consisting of: (i)a quadrupole mass filter; (ii) a 2D or linear quadrupole ion trap; (iii)a Paul or 3D quadrupole ion trap; (iv) a Penning ion trap; (v) an iontrap; (vi) a magnetic sector mass filter; (vii) a Time of Flight massfilter; and (viii) a Wien filter; and/or

(k) a device or ion gate for pulsing ions; and/or

(l) a device for converting a substantially continuous ion beam into apulsed ion beam.

The mass spectrometer may further comprise either:

(i) a C-trap and an Orbitrap® mass analyser comprising an outerbarrel-like electrode and a coaxial inner spindle-like electrode,wherein in a first mode of operation ions are transmitted to the C-trapand are then injected into the Orbitrap® mass analyser and wherein in asecond mode of operation ions are transmitted to the C-trap and then toa collision cell or Electron Transfer Dissociation device wherein atleast some ions are fragmented into fragment ions, and wherein thefragment ions are then transmitted to the C-trap before being injectedinto the Orbitrap® mass analyser; and/or

(ii) a stacked ring ion guide comprising a plurality of electrodes eachhaving an aperture through which ions are transmitted in use and whereinthe spacing of the electrodes increases along the length of the ionpath, and wherein the apertures in the electrodes in an upstream sectionof the ion guide have a first diameter and wherein the apertures in theelectrodes in a downstream section of the ion guide have a seconddiameter which is smaller than the first diameter, and wherein oppositephases of an AC or RF voltage are applied, in use, to successiveelectrodes.

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments of the present invention will now be described, byway of example only, and with reference to the accompanying drawings inwhich:

FIG. 1 shows an impactor spray API source according to an embodiment ofthe present invention; and

FIG. 2A shows an impactor spray source and FIG. 2B shows an optimisedimpactor spray source.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENT

FIG. 1 is a schematic of the general layout of an impactor spray APIsource according to a preferred embodiment. A flow of liquid from a CEcolumn outlet (or other separation device) enters a nebuliser probe 1and is delivered to a sprayer tip 2 via an inner capillary tube 3. Theinner capillary 3 is surrounded by a second concentric capillary 4 whichdelivers a make-up flow of liquid which mixes with the flow from thefirst capillary 3 at the probe tip. The second capillary tube 4 issurrounded by a third concentric capillary 5 which includes a gas inlet6 to deliver a stream of high velocity gas to the exit of the liquidcapillaries 3,4.

This arrangement produces a nebulised spray which contains droplets witha typical diameter of 10-20 μm and velocities greater than 100 m/s at aclose distance from the sprayer tip 2. The resulting droplets are heatedby an additional flow of gas that enters a concentric annular heater 7via a second gas inlet 8.

The sprayer is preferably hinged to the right hand side of an ion inletcone 9 of the mass spectrometer and can swing to vary the horizontaldistance between the sprayer tip 2 and an ion inlet orifice 10 of a massspectrometer. The probe is also configured such that the verticaldistance between the sprayer tip 2 and the ion inlet orifice 10 can bevaried. The relative tip positions of the inner capillary 3, the secondcapillary 4 and the third capillary 5 can be adjusted. According to anembodiment the capillaries 3,4,5 may be arranged so that they are flushwith one another. According to another embodiment the capillaries 3,4,5may be arranged so that one or more capillaries 3,4,5 protrude or arerecessed relative to each other.

A target 11 with a similar dimension to the liquid capillary ispreferably placed between the sprayer tip 2 and the ion inlet orifice10. The target 11 can be manipulated in the x and y directions (in thehorizontal plane) via a micro adjuster stage and can be held at apotential of typically 0-5 kV relative to the source enclosure 12 andthe ion inlet orifice 10. In operation, the ion inlet cone 10 issurrounded by a metal cone gas housing 13 that is flushed with a lowflow of nitrogen gas that enters via a gas inlet 14. All gasses thatenter the source enclosure must leave via the source enclosure exhaust15 or the ion inlet orifice 10 which is pumped by the first vacuum stage16 of the mass spectrometer.

FIG. 2A is a schematic plan view of an impactor spray source with thegrounded nebuliser probe omitted from the diagram. The impactor target11 comprises a stainless steel rod or pin with an outside diameter oftypically 1-2 mm. The rod or pin 11 is positioned at a horizontaldistance X₁ of typically 5 mm from the ion inlet orifice 10. The probetip can be finely adjusted to sweep across the target surface until theoptimum impact point is found that gives the greatest sensitivity. Atypical optimized position is shown in the schematic of FIG. 2B wherethe offset X₂ is approximately 0.4 mm.

FIG. 2B also shows the vertical positions of the probe and target in thepreferred embodiment, i.e. Z₁=9 mm and Z₂=3 mm.

In the preferred embodiment, the source is operated with the followingbias potentials: nebuliser=0V, impactor target=1.0 kV, ion inletcone=100 V and cone gas housing=100 V. The heater assembly and sourceenclosure are preferably maintained at ground potential. The source maybe operated with the following gas flow settings: nitrogen nebulizer gaspressurized to 7 bar, nitrogen heater gas flow=1200 L/hr and nitrogencone gas flow=150 L/hr.

The preferred embodiment can be used in other applications that aresimilarly simplified by the use of a grounded nebuliser probe such ascapillary electrochromatography (CEC) and tile-based microchip LC/MSsystems.

The tile-based microchip LC system preferably comprises a substantiallyrigid ceramic-based multilayer microfluidic substrate also referred toas a “ceramic tile”. Reference is made to US 2009/032135 the contents ofwhich are incorporated herein by reference. For a protein sample theceramic may comprise a High-Temperature Co-fired Ceramic (HTCC) whichprovides suitably low levels of loss of sample due to attachment ofsample to walls of conduits in the substrate. Formed in the layers ofthe substrate is a channel that operates as a separation column.Apertures in the side of the substrate provide openings into the channelthrough which fluid may be introduced into the column. Fluid passesthrough the apertures under high pressure and flows toward theElectrospray emitter coupled at the egress end of the channel. Holes inthe side of a microfluidic cartridge provide fluidic inlet ports fordelivering the fluid to the substrate. Each fluidic inlet port alignswith and encircles one of the fluidic apertures.

The preferred embodiment may also be implemented as an interface forsupercritical fluid chromatography/MS.

Impaction-based spray using a target pin has been shown to provideimproved ionization efficiency for both polar and non-polar compoundscompared to standard ESI or APCI. However, the performance withdifferent mobile phase compositions has sometimes been observed to havea reasonably strong dependence upon the physical geometry of the probeand pin.

The positional dependence of the probe and pin on the relativeperformance at high organic mobile phase can make achieving requiredtolerances problematic. Furthermore, maintaining these tolerances canalso be problematic since the pin and/or probe capillary may need to bereplaced one or more times during the lifetime of the instrument.

According to an embodiment of the present invention a grid or meshtarget is preferably used instead of a pin target. A grid or mesh targethaving a grid or mesh impaction surface has been found to beparticularly advantageous compared with using a pin target in thatutilising a grid or mesh target solves the problem of positionaldependence which may otherwise be experienced when using a solid pin asthe target.

A mesh or grid target of appropriate size is preferably used as theimpact target. According to the preferred embodiment the impact zone(i.e. the diameter of the plume at point of impact with the target) ispreferably 0.5-1.0 mm.

According to the preferred embodiment the mesh wire size and spacing ispreferably sized appropriately so as to provide several discrete impactzones within the impact zone or area. The wire diameter is preferablysufficient so as to allow the impact of the plume on the wire to improvenebulisation. A mesh with 150 μm spacing and a wire diameter of 100 μmhas been found to be particularly advantageous. However, other aspectratios are also contemplated and are intended to fall within the scopeof the present invention. According to an embodiment the mesh or gridmay comprise a substantially flat rectangle (15 mm×7 mm) and may be heldsubstantially perpendicular to the spray axis. According to thisembodiment the spray is essentially through the mesh or grid.

Alternatively, the mesh or grid may be angled relative to the sprayaxis. The angle of the mesh or grid may be set such that the plume as itpasses through the mesh or grid is deflected close to or in thedirection of the mass spectrometer inlet. The mesh or grid target may bearranged at an angle of 70° relative to the spray axis.

The physical dimensions of the mesh or grid are preferably set orarranged so that liquid beading on the surface of the mesh or grid ispreferably minimized. The angle and shape of the mesh or grid may beoptimised to reduce liquid beading.

According to the preferred embodiment a high voltage may be applied tothe mesh or grid electrode in order to assist ionization in a similarmanner to other embodiments of the present invention which have beendescribed above and which utilise a pin target. According to anembodiment the mesh or grid may be maintained at a potential of 1 kV.However, it will be apparent to those skilled in the art that the meshor grid target may be maintained at other potentials.

A particular advantage of using a mesh or grid target is that the meshor grid target according to the preferred embodiment shows asignificantly reduced dependence on positional geometry since the streamof droplets impacts upon multiple impaction points on the mesh or gridtarget. As the probe or mesh target is moved, the characteristics of theimpact of the droplets upon the target remain substantially the same.Accordingly, the performance of the ion source relative to the positionof the MS inlet and the probe behaves in a similar manner to anElectrospray ionisation (“ESI”) ion source relative to an ion inlet.

Further embodiments are also contemplated. For example, a grid insteadof a mesh may be used. The grid preferably has multiple impaction pointsin the zone in which the stream of droplets impacts upon the target. Ifpositional dependence of the spray direction after impact is requiredthen a single-row grid may be utilised.

According to an embodiment the target may comprise multiple layers ofmeshes and/or grids in order to achieve the same effect as angling asingle layered mesh or grid target.

According to an embodiment the surface ionization impactor bar or targetas described above may be further enhanced by utilising a piezoelectricvibration device to vibrate the bar or target. Vibration of the bar ortarget upon which the surface ionization occurs aids in the reduction ofthe size of the secondary droplets, increasing the evaporation rate ofthe solvent and thereby aids signal response.

According to a preferred embodiment an impactor bar or target is locatedwithin a source enclosure. In this configuration the capillary ispreferably grounded and potentials are preferably applied to theimpactor bar or target and to the sample cone inlet structure. Theintegration of an impactor spray with a separation device introduces thepotential for the generation of non-polar, highly polar, singularlycharged and/or multiply charged gas phase ions for introduction into themass spectrometer for analysis. The ionization processes and flowdynamics may, however, be different which can result in the formation oflarger sized droplets. The use of piezoelectric vibration applied to theimpactor bar or target is particularly advantageous in that it aids inthe reduction of resultant secondary droplets.

It will be understood by those skilled in the art that the mechanisms ofdroplet production in pneumatically assisted nebulisation arenon-trivial and it cannot be approximated by a particular model forwhich the boundary conditions are known. There is no single process thatis believed to be solely responsible for droplet production and theinitial spray produced is rapidly modified by secondary fragmentationand by recombination and coalescence. The use of piezoelectric vibrationapplied to the impactor bar or target preferably aids in the reductionof the resultant secondary droplets through surface disruption.

According to an embodiment a target pin is preferably utilised which ispreferably rotated on e.g. an eccentric path so as to obtain an easilyreproducible level of ion signal. According to an embodiment a targetpin or rod is preferably placed or mounted off axis on a rotating shaft.The pin or rod target is preferably located or arranged so as to be inthe path of high velocity droplets emitted from a sprayer. The dropletsemitted from the sprayer are arranged to impact upon the pin or rodtarget so as to produce ions for analysis by mass spectrometry. Therotational position of the pin or rod is preferably controlled through amotor under computer control.

According to an embodiment the analyte signal may be monitored withrespect to the position of the pin or rod. The pin or rod may then berotated or otherwise set to a particular position under computer controlin order to maximise the signal intensity. Other embodiments are alsocontemplated wherein the pin or rod may be rotated between one or moredifferent rotational positions in order to control the intensity ofanalyte ions produced or to control the efficiency of analyte ionproduction.

The central longitudinal axis of the pin or rod is preferably arrangedso as to be off centre relative to the central longitudinal axis of therotating shaft. The position of the pin or rod may according to anembodiment vary by approximately 0.7 mm during the course of onerotation of the pin or rod target.

According to a less preferred embodiment the position of the pin or rodtarget 10 may be translated rather than rotated (or the pin or rodtarget may be translated in addition to being rotated).

As will be understood by those skilled in the art the positioning of thetarget is important in order to obtain an acceptable level of signalintensity when generating ions by impacting high velocity droplets ontothe target. According to a particularly preferred embodiment causing thetarget to rotate on an eccentric path relative to the spray of highvelocity droplets enables an average signal intensity to be realised. Asa result, the overall or average ion signal can be stabilised and isless susceptible to wide variations in the intensity of analyte ionsgenerated depending upon the precise position of the target relative tothe high velocity spray of droplets.

Although the present invention has been described with reference topreferred embodiments, it will be understood by those skilled in the artthat various changes in form and detail may be made without departingfrom the scope of the invention as set forth in the accompanying claims.

The invention claimed is:
 1. A mass spectrometer comprising: aseparation device arranged and adapted to emit an eluent over a periodof time, wherein said separation device comprises either: (i) aCapillary Electrophoresis (“CE”) separation device; (ii) a CapillaryElectrochromatography (“CEC”) separation device; (iii) a substantiallyrigid ceramic-based multilayer microfluidic substrate (“ceramic tile”)separation device; or (iv) a supercritical fluid chromatographyseparation device; a nebuliser and; a target; wherein said eluentemitted by said separation device is nebulised, in use, by saidnebuliser wherein a stream of analyte droplets are directed to impactupon said target so as to ionise said analyte to form a plurality ofanalyte ions.
 2. A mass spectrometer as claimed in claim 1, wherein thesubstantially rigid ceramic-based multilayer microfluidic substrateseparation device comprises a High-Temperature Co-fired Ceramic(“HTCC”).
 3. A mass spectrometer as claimed in claim 1, wherein thenebuliser is constructed with a tri-axial probe arrangement.
 4. A massspectrometer as claimed in claim 1, wherein said separation devicecomprises or is coupled to a first tube.
 5. A mass spectrometer asclaimed in claim 4, wherein said first tube comprises a capillary tube.6. A mass spectrometer as claimed in claim 4, wherein said first tube issurrounded by a second tube which is arranged and adapted to provide aflow of liquid which mixes with the eluent emerging from the exit ofsaid first tube.
 7. A mass spectrometer as claimed in claim 6, whereinsaid second tube comprises a capillary tube.
 8. A mass spectrometer asclaimed in claim 7, further comprising a third tube which is arrangedand adapted to provide a stream of gas to the exit of said first tube orsaid second tube.
 9. A mass spectrometer as claimed in claim 8, whereinsaid third tube comprises a capillary tube.
 10. A mass spectrometer asclaimed in claim 8, wherein said third tube surrounds said second tubeor is concentric with said first and second tubes.
 11. A massspectrometer as claimed in claim 8, wherein said third tube isnon-concentric with said first and said second tubes.
 12. A massspectrometer as claimed in claim 1, wherein said target comprises one ormore mesh or grid targets.
 13. A method of mass spectrometry comprising:providing a separation device arranged and adapted to emit an eluentover a period of time, wherein said separation device comprises either:(i) a Capillary Electrophoresis (“CE”) separation device; (ii) aCapillary Electrochromatography (“CEC”) separation device; (iii) asubstantially rigid ceramic-based multilayer microfluidic substrate(“ceramic tile”) separation device; or (iv) a supercritical fluidchromatography separation device; providing a target; and nebulisingsaid eluent emitted by said separation device wherein a stream ofanalyte droplets are directed to impact upon said target so as to ionisesaid analyte to form a plurality of analyte ions.
 14. A method of massspectrometry as claimed in claim 13, further comprising delivering amake-up flow of liquid that mixes with the liquid flow from theseparation device.
 15. A method of mass spectrometry as claimed in claim13, wherein a flow of liquid from the separation device enters anebuliser probe and is delivered to a sprayer tip via a first tube. 16.A method of mass spectrometry as claimed in claim 15, wherein the firsttube is surrounded by a second tube which delivers a make-up flow ofliquid which mixes with the flow from the first tube at the probe tip.17. A method of mass spectrometry as claimed in claim 16, wherein thesecond tube is surrounded by a third tube which includes a gas inlet todeliver a stream of high velocity gas to the exit of the first andsecond tubes.
 18. A mass spectrometer comprising: a separation devicearranged and adapted to emit an eluent over a period of time; anebuliser and; a target; wherein said eluent emitted by said separationdevice is nebulised, in use, by said nebuliser wherein a stream ofanalyte droplets are directed to impact upon said target so as to ionisesaid analyte to form a plurality of analyte ions; and wherein said massspectrometer further comprises a control system, wherein said controlsystem is arranged and adapted either: (i) to switch the polarity ofsaid target during a single experimental run; or (ii) to repeatedlyswitch the polarity of said target during a single experimental run. 19.A method of mass spectrometry comprising: providing a separation devicearranged and adapted to emit an eluent over a period of time; providinga target; and nebulising said eluent emitted by said separation devicewherein a stream of analyte droplets are directed to impact upon saidtarget so as to ionise said analyte to form a plurality of analyte ions;wherein said method further comprises providing a control system,wherein said control system is arranged and adapted either: (i) toswitch the polarity of said target during a single experimental run; or(ii) to repeatedly switch the polarity of said target during a singleexperimental run.